Preparation Of Goat Fetal Fibroblast Cell Lines For Nuclear Transfer To Express RHSA In The Chimeric HLA-YAC

Transgenesis is commonly applied to study gene function, to devise new animal models of human genetic diseases or to produce recombinant proteins in milk of transgenic animals. However, there is still a major limitation in methodology, namely, the uncertainty about the expression of each transgene. This is mainly caused by the stochastic event of transgene integration within the host genome and the nature of the transgenic constructs. One of the main strategies proposed to overcome these position effects is genomic fragment with a given expression domain as transgenic constructs. Such conditions are present in yeast artificial chromosome vector due to its large cloning capacity. Genomic large fragments cloned in yeast artificial chromosome vector can guarantee optimal expression levels in transgenic animals regardless of the positions of integration. Meanwhile, it is characteristic of position-independent, copy number-dependent and tissue-specific transgenic expression, and their expression is comparable to that of endogenous levels in most cases. The object of this study is to express human serum albumin (HS A) in the mammary gland of transgenic goat from human α-lactalbumin (ha-Lac) YAC (HLA-YAC) of CEPH library.The CEPH library was constructed in the host strain AB1380. The genetic markers in this strain are of limited utility in homologous recombination manipulations. Thus, the need to move YACs into a genetic background that will accommodate a desired modification is commonly encountered. In virtue of the properties of karl mutants, YPH925 loses the ability of nuclear fusion when it mates with opposite mating type strain. The HLA-YAC is transferred from donor yeast strain AB1380 to recipient YPH925 with genetic markers that facilitate the use of existing homologous recombination-based modification methods. These clones were analyzed by pulsed-field gel electrophoresis and PCR. They showed HLA-YAC was transferred to YPH925 strain. Two different markers were used to explore the feasibility of specific-site mutants to HLA-YAC in yeast. Two homologous arms cloned from HLA-YAC by PCR, 940 bp and 680 bp from 5' and 3' untranslated region of a-lactalbumin gene, respectively, and a yeast dominant selectable marker (G418R) were placed in the pRS403 containing HIS3 as the targeting vector (pLAG418R). Linearized targeting vector is introduced into yeasts by transformation with the methods of lithium acetate or electroporation. The clones are analyzed using PCR, as well as sequencing. The results showed the HLA-YAC is modified by homologous recombination. The efficiency of G418R selection is significantly higher.than HIS3, and positive efficiency by lithium acetate transformation is a little higher than that by electroporation.A targeting vector was constructed to use the regulatory elements of cc-lactalbumin gene on the HLA-YAC to direct the expression of HSA in the mammary gland of transgenic animals. A mini-rHSA containing intron 1 -2, and neor from pcDNA3 were cloned in the downstream of 5' homologous arm of pLAG418. Linearized targeting vector is introduced into yeasts by transformation with the methods of lithium acetate. The clones are analyzed using PCR, as well as Southern blot. They showed the HLA-YAC is modified by homologous recombination. A chimeric YAC to express rHSA of the mammary gland in the transgenic animals was contracted.Microinjection of yeast artificial chromosomes (YACs) is the exclusive technique used to produce YACs transgenic livestock at present. However, its lower efficiency is a restrictive factor. In addition to intensive labor and tiresome manipulation on the isolation of YACs from yeast, the low integration efficiency (5%) and few intact YACs (20-70%) in the organism genome by microinjection are large obstacles to produce YACs transgenic livestock. In this study, we presented a novel procedure to produce YACs transgenic livestock as mammary gland bioreactor. The chimeric YACs were introduced into goat fetal fibroblasts using polyethylene glycol (PEG)-mediated spheroplast fusion...